Unusual secondary structures in linear DNA fragments often lead to significant conformational changes of the entire molecule. Such conformational changes of linear nucleic acids fragments frequently result in curvature, characterized by decreased end-to-end distance. Curvature has for example been detected or proposed in linear DNA fragments containing mispaired bases, insertion/deletion loops, UV-lesions, base adducts, base methylation, A-tracts, GGCC sequences, cross-links, DNA hairpins or cruciforms, slipped-strand structures, protein binding and nicking. DNA conformation in general, and conformational changes of linear DNA fragments because of altered secondary structure, have been analyzed using various direct and indirect methods such as X-ray diffraction, nucleic magnetic resonance spectroscopy (NMR), electron microscopy (EM), fluorescence resonance energy transfer (FRET), and gel retardation analysis.
Some gel electrophoresis methods allow separation of linear DNA fragments based on both length and conformation, while others separate DNA fragments based essentially on their length. Polyacrylamide gel electrophoresis (PAGE) is an example of a method in the former category and agarose electrophoresis an example of the latter. A rigorous physical theory of gel electrophoresis through a matrix capable of separating DNA fragments based on length, or both length and conformation, has not been presented. However, it has been recognized that PAGE allows conformation-dependent separation of linear DNA fragments in such way that migration decreases as the square of the degree of curvature for each linear DNA fragment separated [1]. This quantitative relationship between curvature and migration of linear DNA fragments has been used experimentally to evaluate DNA curving. Such methods are based on the fact that linear DNA fragments with altered conformation migrate at a different rate through a gel matrix compared to DNA fragments with normal conformation. These differences in migration rates can further be used to separate and isolate linear DNA fragments based on their conformation if the length of each fragment is known. In principle, it should be possible to separate linear DNA fragments of equal length with various conformations using PAGE with sufficient resolution.
A few previously described analytical methods used to screen for mutations or polymorphism are based on this principle. Heteroduplex analysis (HA) is a method used to scan for polymorphism by comparing migration rates of mismatched heterohybrids and perfectly matched hetero- and homohybrids. After melting and reannealing of homologous DNA samples from two or more individuals, the DNA mixture will contain both heterohybrids, some or all are mismatched if there is polymorphism between individuals, and perfectly matched homohybrids. Alternatively, the mixture will contain perfectly matched hetero- and homohybrids if the DNA samples are identical in sequence. After reannealing, the DNA mixture is analyzed using PAGE. If the sample contains mismatched heterohybrids, their migration will be retarded through the gel due to their altered conformation compared to the perfectly matched duplexes. Several variations to increase the sensitivity and reliability of HA have been developed. Conformation Sensitive Gel Electrophoresis (CSGE) is a well-known variation of HA [2, 3]. This system uses mildly denaturing solvents to enhance the tendency of single-base mismatches to produce conformational changes (see U.S. Pat. No. 5,874,212 to Prockop, et al.).
Use of previously described methods for conformation-dependent separation of linear DNA fragments, such as HA and CSGE, are limited to situations were the analyzed DNA molecules are of known length. In addition, only simple DNA samples containing one or at most a few different DNA fragments can be tested. If the sample contains a complex mixture of DNA fragments of different lengths it would be impossible to identify which of them show difference in migration because of their conformation since individual bands would overlap or not resolve sufficiently. This is a major drawback since it limits application of this technology. Similar limitations would also apply to other comparable techniques based on capillary electrophoresis or chromatography (e.g. dHPLC).
Methods for separating individual linear DNA fragments from a complex mixture based only on their difference in conformation, independent of their length, would be of great interest. Such methods would allow analysis of complex samples containing many linear DNA fragments of different length. Examples where such methods could be used include but are not limited to: Physical separation of mismatched heterohybrids and perfectly matched hetero- and homohybrids allowing isolation and enrichment of either class; simultaneous mismatch scanning of multiple fragments; isolation of damaged DNA molecules from bulk amount of undamaged molecules, and estimation of the efficiency of nucleic acid reannealing.
One possible way to achieve length independent, or essentially length independent, separation of linear DNA fragments based on their conformation is to develop two-dimensional (2-D) gel electrophoresis systems. Such a system would separate DNA fragments based on both length and conformation in one dimension but only on length in the other dimension.
Two different 2-D gel electrophoresis methods capable of separating linear DNA fragments based on conformation were described in the late eighties [4, 5]. These two methods use different approaches to resolve linear DNA fragments with certain conformations. Both of these methods provided separation of curved linear DNA fragments containing adenine-tracts (A-tracts). One of these methods combines agarose and PAGE electrophoresis using the different migration behavior of curved linear DNA fragments in these two matrixes. The other method uses temperature-dependency of DNA structure and conformation. By using different temperature for each dimension (10° C. and 60° C.) different conformations are induced resulting in differences in migration rates. DNA curvature due to A-tracts results from different stacking interactions between adjacent adenine bases in the molecule. A-tract curvature is not very rigid and can therefore be removed or reduced by increasing the temperature of the system [6, 7]. It is not disclosed or suggested in the prior art, that these or other systems can separate other more rigid conformations, such as those formed in mismatched or UV damaged duplexes. In our experience, it is not possible to separate mismatched duplexes from perfectly matched duplexes using temperature as a variable between the two dimensions. This is perhaps due to secondary and tertiary structures resulting from mismatches are less temperature dependent than in the case of A-tracts. The combined agarose-PAGE system is limited by difficulties in transferring linear DNA fragments between the two different matrixes in an efficient and reproducible manner.
A 2-D gel electrophoresis system using neutral-neutral agarose gel electrophoresis for the separation of relaxed circular DNA and supercoiled DNA from linear DNA using ethidum bromide in the second dimension is disclosed in WO 97/39149. In the first dimension, DNA molecules are separated in proportion to their mass using low voltage in low percentage agarose. The second dimension is run at high voltage in a gel of higher agarose concentration in the presence of the intercalator ethidium bromide. Under these conditions, mobility of all circular DNA molecules is drastically influenced by their shape but mobility of linear DNA fragments are essentially the same as in the first dimension. After separation and nucleic acid staining a pattern is detected. The pattern consist of generally three arcs lying in front of the forth arc which contains linear or linearized DNA. Arc 1 contains opened circles (relaxed) DNA. Arc 2 contains covalently closed circles that were converted to a relaxed form. Arc 3 contains covalently-closed (supercoiled) DNA. It should be emphasized that this method cannot be used to separate linear DNA fragments according to conformation because of the fact that linear DNA fragment of same length with different conformation are separated almost entirely according their size in agarose gel electrophoresis. Further, perhaps due to these limitations, the reference does not disclose or suggest 2-D gel electrophoresis methods for separating other types of conformational different DNA molecules, such as curved linear DNA fragments containing unusual secondary structure, e.g. mismatched duplexes.
Two types of 2-D gel electrophoresis system have been described for mapping origins of replication [8, 9]. One of the systems uses neutral-neutral agarose gel electrophoresis as described above. The other system uses neutral-alkaline agarose gel electrophoresis to achieve separation according to size and structure in the first dimension and only size in the second dimension. These systems allow separation of large non-circular DNA molecules with unusual structure such as those formed in replicons (large DNA bubbles and Y shaped DNA replication forks). Linear DNA fragments that are curved due to existence of local unusual secondary structure such as UV-lesion or insertion bulges cannot be separated in these systems. The difference in conformation of such linear DNA fragment with or without unusual local secondary structures is not great enough to ensure different migration in agarose elctrophoresis and will therefore not be detected in this system.
An ideal 2-D gel electrophoresis system for conformation-dependent separation of linear nucleic acids fragments would preferably be based on a single gel matrix eliminating the troublesome transfer step between two different gel matrixes. Such transfer often gives rise to trailing effects, which lower the detection capability of the system. A physical or chemical factor would then be introduced (or removed) after the first dimension to affect the conformation of different linear DNA fragments to a different degree depending on secondary and tertiary structure. Such physical or chemical factor should allow the differentiation of minor as well as major conformational differences, such as e.g. caused by single base pair mismatched DNA and insertion or deletion bulges.
Many chemical factors have been reported to affect conformation of linear DNA molecules, e.g. mono- and divalent cations such as Na+ and Mg2+. DNA curvature generally increases with increasing concentration of cations. Chemical factors that decrease curvature of DNA fragments containing unusual secondary structures would generally be of more interest than those that increase DNA curvature. Intercalators are small planar molecules, which form hydrophobic interactions with nucleic acids by insertion (intercalation) between DNA base pairs. Such interactions require untwisting of the DNA molecule to enable enough space between adjacent base pairs for the intercalator molecule. This separation of base pairs and concurrent untwisting results in increased length and stiffness of the DNA molecule thus affecting its whole conformation [10]. It has been reported that addition of intercalators to linear DNA fragments, curved because of A-tract, can greatly reduce the curvature [11-14]. This has been determined both with PAGE electrophoresis and electron microscopy studies. We have now defined conditions were some intercalators also greatly reduce the curvature of linear DNA fragments containing insertion/deletion loops or UV adducts as determined by gel retardation analysis in PAGE electrophoresis.
It is a well-known fact that linear single-stranded DNA fragments migrate essentially only according to length in PAGE containing highly concentrated denaturating chemical agents. Such behavior of single-stranded DNA is the basis of common techniques in DNA sequencing. We have found that after separation of mismatched DNA molecules both according to length and conformation it is possible to denaturate DNA molecules in the gel with addition of denaturating chemical agents and heating for given amount of time. Thus, it is possible to separate single stranded DNA fragments in the second dimension only according to length allowing formation of electrophoresis system capable of conformation dependent separation.